This method provides a new approach to addressing key questions in the field of pulmonary vascular research, such as the molecular path of mechanisms of intimal proliferation in pulmonary hypertension. The main advantage of this method is that it provides a minute cellular biopsy of the pulmonary vascular endothelium without additional risk to the patient. Demonstrating the procedure will be Miss Reina Perez, our Clinical Coordinator, and Dr.Phillip Gallo, the manager of my laboratory.
After the catheter is withdrawn from the sheath, immediately immerse it in a 50 milliliter centrifuge tube containing 40 milliliters of chilled sterile saline. Swirl the catheter several times to remove any blood, and flush the line if necessary to remove the blood retained within it. Cut off the distal five centimeters of the catheter tip, and allow it to fall into the centrifuge tube.
Use forceps to immediately remove the tip, and transfer it to a 15 milliliter centrifuge tube containing 15 milliliters of endothelial cell-growth medium. Next, seal the tube. Place the tube on ice, and transfer it to the lab.
Either process the sample immediately or pack the sample on wet ice for overnight shipping to the laboratory where processing will occur. It is critical that the balloon stays immersed in the medium. So make sure there is minimal head space in the 15 mL tube by filling it completely with endothelial cell-growth medium.
First, add five milliliters of cell detachment solution to a clean 15 milliliter centrifuge tube. Place this tube on ice. Using a one milliliter syringe, draw one milliliter of endothelial cell media from what remains in the bedside collection tube.
Carefully insert the needle of the one milliliter syringe into the white hole opposite of the guide wire. Place the catheter tip into the 15 milliliter centrifuge tube that contains the cell detachment solution. Inflate the balloon with approximately 200 microliters of media, being careful not to burst it.
Place the tube on ice, and let the catheter sit in the cell detachment solution for five minutes with the balloon inflated. After this, pour the contents of the tube into a 100 millimeter dish, making sure that the balloon is placed in this puddle of cell detachment solution. Using a cell scraper, scrape the inflated balloon, focus on the poles of the balloon where it meets the catheter.
Next, make sure the cell scraper contacts the entire surface of the balloon at least twice, and periodically rinse the cell scraper in the cell detachment solution. Deflate the balloon and scrape it with a brushing motion. Then rinse the cell scraper in the puddle of cell detachment solution.
Discard the catheter tip, the needle, and the cell scraper into an appropriate waste container. Pour the cell detachment solution that contains the cell harvest into a 50 milliliter centrifuge tube. Shake the collection tube, and pour the remaining media from it into the collected sample as well.
Centrifuge the sample at 650 times G, and four degrees Celsius for 10 minutes. Aspirate the media, being careful to not disturb or aspirate the pellet. Resuspend the pellet in one milliiter of chilled red blood cell lysis solution, and transfer this suspension to a 1.5 microliter centrifuge tube, and gently rock the tube at four degrees celsius for 10 minutes.
Centrifuge the tube at 500 times G, and four degrees celsius for five minutes. Then aspirate the red blood cell lysis solution. Resuspend the pellet in a solution containing PBS, FcR blocker, and anti-human CD-146 microbeads.
Incubate at four degrees celsius for 15 minutes. Add one milliliter of chilled PBS. Centrifuge at 500 times G and four degrees celsius for five minutes.
Aspirate the supernatant and resuspend the pellet in one milliliter of chilled PBS. Next, place a magnetic LS column on a magnetic rack at room temperature. Prime the column by adding one milliliter of PBS, and allowing the entire volume to flow through.
Discard the flow-through. Load the sample on the magnetic column, and discard the flow-through. Wash the column three times using three milliliters of chilled PBS for each wash.
Discard all of this flow-through. Then add three milliliters of chilled PBS to a 15 milliliter centrifuge tube. Remove the magnet column from the rack, and pour the PBS from the tube into the column.
Quickly place the column into the centrifuge tube to catch the flow-through. Immediately plunge with the plunger supplied with the column. The sample is now purified, and ready for fixation, staining, or culture.
In this study, cells are purified from Swan-Ganz catheter tips after right heart catheterization. These cells bind to a CD-146 selection column, and have forward and side scatter profiles that are indistinguishable from cultured primary human pulmonary artery endothelial cells. As the catheter balloon is only in physical contact with the introducer sheath, the pulmonary artery vessel wall and the blood, and the cells are demonstrated to not derive from the blood, it is therefore assumed that they derived from the pulmonary artery vessel wall.
Following the procedure harvested cells can be analyzed by a variety of methods, such as flow cytometry, qPCR, sequencing, and proteomix. Cell yields are too low for conventional Western blot, but low input blotting methodologies might be considered. Cells can be cultured, but this is very difficult, and it doesn't always succeed in our hands.
If culture is to be attempted, it's important to minimize the interval between the collection of the cath tip from the patient, and the final purification and plating steps, and to insure that the sample is kept cold throughout all handling steps of the method. Don't forget that this protocol involves working with human blood and tissue. Universal precautions must be observed.
In particular, there's a step early in the protocol that involves inserting a sharp needle into the cut end of the catheter tip which is contaminated with the patient's blood. Never forget that the catheter tip must be held with hemostats during this step, not with your fingers, in order to avoid the risk of a needle stick.
